Liquid formulation of protein conjugate comprising the oxyntomodulin and an immunoglobulin fragment

ABSTRACT

Disclosed are an albumin-free liquid formulation including a long-lasting oxyntomodulin conjugate in which an oxyntomodulin peptide comprising a derivative, variant, precursor or fragment of oxyntomodulin is linked to an immunoglobulin Fc region, which can increase the duration of physiological activity of the long-lasting oxyntomodulin conjugate and maintain the in vivo stability thereof for an extended period of time, as compared to native oxyntomodulin, and a method for preparing the liquid formulation. The liquid formulation contains a buffer, a sugar alcohol and a nonionic surfactant and does not contain a human serum albumin and factors that are potentially harmful to the human body, and thus is not susceptible to viral infection. The oxyntomodulin conjugate contains oxyntomodulin linked to an immunoglobulin Fc region, and thus has a large molecular weight, prolonged physiological activity, and excellent storage stability, compared to native oxyntomodulin.

TECHNICAL FIELD

The present invention relates to an albumin-free liquid formulationcomprising a long-lasting oxyntomodulin conjugate in which anoxyntomodulin peptide comprising a derivative, variant, precursor orfragment of oxyntomodulin is linked to an immunoglobulin Fc region,which can increase the duration of physiological activity of thelong-lasting oxyntomodulin conjugate and maintain the in vivo stabilitythereof for an extended period of time, compared to nativeoxyntomodulin. The present invention also relates to a method forpreparing the liquid formulation.

BACKGROUND ART

Obesity is defined as a condition of abnormal or excessive fataccumulation that may impair health and results from an energy imbalancein which energy intake exceeds energy expenditure. Obesity was not aserious health problem in the past, but with economic growth, the obesepopulation is increasing with increasing economic affluence, and thenumber of various diseases related to obesity is also increasing.According to the report of the World Health Organization (WHO), over 1.5billion adults worldwide are overweight, over 500 million of them areobese, and the obesity population increased by about twice between 1980and 2008 (World Health Organization, Fact sheet on obesity andoverweight, 2011). Not only in high-income countries, but also inlow-income countries, the percentage of obese people is currentlyincreasing. Overweight and obesity are responsible for increasing bloodpressure and cholesterol levels and cause or worsen various diseases. Inaddition, the obesity problem is more serious in children or teenagers,increases the incidence of diabetes, heart diseases, hypertension orhyperlipidemia, and can also cause deaths or disabilities.

As described above, obesity is a global disease and social problem, butin the past, it was believed that obesity could be overcome byindividual efforts, and thus no particular emphasis was placed on thetreatment of obesity. However, obesity is not easy to treat, because itis a complex disease associated with the mechanisms of appetite controland energy metabolism. Accordingly, the treatment of obesity requiresnot only the patient's own efforts, but also a method capable oftreating abnormal mechanisms associated with appetite control and energymetabolism. Thus, efforts have been made to develop drugs for treatingobesity.

As a result of such efforts, drugs, including Rimonabant(Sanofi-Aventis), Sibutramine (Abbott), Contrave (Takeda), Orlistat(Roche) and the like, were developed, but these drugs have shortcomingsin that they show fetal side effects or have an insufficient effect onthe treatment of obesity. It was reported that Rimonabant(Sanofi-Aventis) showed disorders of the central nervous system,Sibutramine (Abbott) and Contrave (Takeda) showed cardiovascular sideeffects, and Orlistat (Roche) showed a weight reduction effect of onlyabout 4 kg when administered for 1 year. Thus, there are currentlylittle or no obesity therapeutic agents that can be safely prescribedfor obesity patients.

Recently, glucagon derivatives have received much attention. Glucagon isproduced by the pancreas when blood glucose levels start to drop due tomedications, diseases, hormone or enzyme deficiencies, or the like.Glucagon functions to stimulate liver cells to break down storedglycogen into glucose which is then released into the blood to raise theblood glucose level to a normal level. In addition to the effect ofincreasing the blood glucose level, glucagon was reported to suppressappetite and activate hormone-sensitive lipase (HSL) of adipocytes tofacilitate lipolysis, thereby showing anti-obesity effects. Among theglucagon derivatives, glucagon-like peptide-1 (GLP-1) is underdevelopment as a therapeutic agent for reducing hyperglycemia indiabetic patients and functions to stimulate insulin synthesis andsecretion, inhibit glucagon secretion, suppress gastric emptying,increase glucose utilization and inhibit food intake. It is known thatexendin-4 that is isolated from lizard venom has an amino acid homologyof about 50% with GLP-1 and activates the GLP-1 receptor to reducehyperglycemia in diabetic patients. However, obesity therapeutic drugs,including GLP-1, were reported to cause side effects such as vomitingand nausea.

Thus, as an alternative to GLP-1, oxyntomodulin capable of binding toboth receptors for two peptides (GLP-1 and glucagon) is receivingattention. Oxyntomodulin is a peptide made from pre-glucagon, aprecursor of glucagon, and is a potent anti-obesity agent, because itinhibits food intake, like GLP-1, promotes satiety, and shows lipolyticactivity, like glucagon.

Based on the dual function of the oxyntomodulin peptide, studies on thedevelopment of drugs for the treatment of obesity have been activelyconducted. For example, Korean Patent Registration No. 925017 disclosesan oral, parenteral, mucosal, rectal, subcutaneous or transdermalpharmaceutical composition for treating human obesity, which comprisesoxyntomodulin as an active ingredient. However, it was reported thatobesity therapeutic agents comprising oxyntomodulin have a short in vivohalf-life and show a low effect on the treatment of obesity, even whenthese are administered at a high dose three times a day. Thus, effortshave been made to increase the in vivo half-life or obesity-treatingeffect of oxyntomodulin by modifying oxyntomodulin.

For example, the dual agonist oxyntomodulin (Merck) is obtained bysubstituting L-serine with D-serine at amino acid 2 of oxyntomodulin toincrease resistance to dipeptidyl peptidase-IV (DPP-IV) and by attachinga cholesterol moiety to the C-terminal to increase the blood half-life.ZP2929 (Zealand) is obtained by substituting L-serine with D-serine atamino acid 2 of oxyntomodulin to increase resistance to DPP-IV,substituting arginine with alanine at amino acid 17 to increaseresistance to protease, substituting methionine with lysine at aminoacid 27 to increase oxidative stability, and substituting glutamines atamino acids 20 and 24 and asparagine at amino acid 28 with asparticacid, alanine and serine, respectively, to increase deamidationstability. The dual agonist oxyntomodulin (Merck) has an increased invivo half-life of 1.7 hours, which is longer than the half-life (8-12minutes) of native oxyntomodulin, but it still has a very short in vivohalf-life and is administered at a very high dose of several mg/kg.Thus, oxyntomodulin or derivatives thereof have two big disadvantages,that is, a short half-life and low medicinal effects. Due to thesedisadvantages, they should be administered daily at high doses. In orderto overcome these disadvantages, a method was studied to increase theblood half-life of oxyntomodulin while maintaining the in vivo activitythereof, and as a result, an oxyntomodulin derivative was developed. Inaddition, using this technology, a non-peptidyl polymer was prepared byconjugating a carrier to the oxyntomodulin derivative, and it was foundthat the protein conjugate can show a better anti-obesity effect as aresult of increasing the blood half-life thereof while maintaining thein vivo activity (Korean Patent Application No. 10-2012-0064110).

Generally, proteins and peptides have a very short half-life, andundergo denaturation such as precipitation by aggregation of monomers,and adsorption on the surfaces of vessels, upon exposure to variousfactors such as unfavorable temperatures, water-air interface, highpressure, physical/mechanical stress, organic solvents and microbialcontamination. This denaturation is irreversible, and thus the denaturedproteins and peptides lose intrinsic physicochemical properties andphysiologically active effects. In addition, proteins and peptides areunstable and susceptible to extrinsic factors such as temperature,humidity, oxygen, UV rays or the like to undergo physical or chemicalchanges including association, polymerization or oxidation, resulting insubstantial loss of activity (Korean Patent Registration No.10-0389726).

Furthermore, the adsorbed proteins and peptides are easily aggregated bythe denaturation process, and the denatured proteins and peptides, whenadministered to the human body, act as the cause of antibody formationin the human body, and for this reason, the proteins and peptides shouldbe administered in a sufficiently stable form. Accordingly, variousmethods for preventing the denaturation of proteins and peptides insolution have been studied (John Geigert, J. Parenteral Sci. Tech., 43,No 5, 220-224, 1989; David Wong, Pharm. Tech. October 34-48, 1997; WeiWang., Int. J. Pharm., 185, 129-188, 1999; Willem Norde, Adv. ColloidInterface Sci., 25, 267-340, 1986; Michelle et al., Int. J. Pharm. 120,179-188, 1995).

Lyophilization is applied to some protein and peptide drugs to achievethe goal of stability. However, lyophilized products are inconvenient inthat they must be re-dissolved in injection water for use. In addition,in the case of lyophilization, massive investment on large-capacityfreeze-driers or the like is required, because the lyophilizationprocess is included in the production processes. Further, a method forproducing powdered proteins and peptides using a spray drier is alsobeing used, but in this case, economic efficiency is decreased due to alow yield, and exposure to high temperatures can adversely affect thestability of the proteins.

In order to overcome such limitations, studies have been conducted inwhich stabilizers were added to proteins and peptides in solution tosuppress the physicochemical changes of the proteins and peptides whilemaintaining the in vivo efficiency thereof even upon long-term storage.Human serum albumin, a kind of protein, has been widely used as astabilizer for various protein drugs, and the performance thereof hasbeen proven (Edward Tarelli et al., Biologicals (1998) 26, 331-346).

A process for purifying human serum albumin includes inactivatingbiological contaminants such as mycoplasma, prion, bacteria and virusand screening or examining one or more biological contaminants orpathogens. However, there is always the risk that patients will beexposed to the biological contaminants that are not completely removedor inactivated. For example, the screening process includes examiningwhether human blood from donators contains a certain virus, but thisprocess is not always reliable. Particularly, a specific virus existingin a very small number of donators cannot be detected.

Different proteins may be gradually inactivated at different rates underdifferent conditions during storage, due to their chemical differences.That is to say, the extension of the storage term by a stabilizer is notidentical for different proteins. For this reason, the suitable ratio,concentration and kind of stabilizer that is used to provide storagestability vary depending on the physicochemical properties of the targetprotein. When stabilizers are used in combination, they may causeadverse effects different from desired effects due to the competitionand interaction therebetween. Further, because the nature orconcentration of proteins may change during storage, the stabilizersused may show effects different from those intended. Thus, a greatamount of effort and precautions are required to stabilize proteins insolution.

Particularly, a conjugate of oxyntomodulin and immunoglobulin Fc is aconjugate in which oxyntomodulin that is a physiologically activepeptide is linked to an immunoglobulin Fc region. Thus, because themolecular weight and volume of the conjugate certainly differ from thoseof native oxyntomodulin, a special composition for stabilizing theprotein is required.

Further, because oxyntomodulin (that is a physiologically activepeptide) and the immunoglobulin Fc region are peptides or proteinshaving different physicochemical properties, they should besimultaneously stabilized. However, as described above, differentproteins or proteins may be gradually inactivated at different ratesunder different conditions during storage, due to their chemicaldifferences, and when stabilizers suitable for proteins or peptides areused in combination, they may cause adverse effects different from thedesired effects due to the competition and interaction therebetween.Thus, in the case of a long-lasting oxyntomodulin conjugate, there ismuch difficulty in finding a composition for simultaneously stabilizingoxyntomodulin, which is a physiologically active peptide, and theimmunoglobulin Fc region.

Under such circumstances, the present inventors have made extensiveefforts to provide a stable liquid formulation that can be stored for along period of time without concern about viral contamination, and as aresult, have found that a stabilizer, which includes a buffer, a sugaralcohol and a nonionic surfactant and may further include an additive,such as an isotonic agent or an amino acid, and a preservative forrepeated use, can increase the stability of a long-lasting oxyntomodulinderivative, and a cost-effective and stable liquid formulation can beprepared using the stabilizer, thereby completing the present invention.

DISCLOSURE Technical Problem

It is an object of the present invention to provide a liquid formulationof a long-lasting oxyntomodulin conjugate, comprising apharmacologically effective amount of a long-lasting oxyntomodulinconjugate wherein an oxyntomodulin which is a physiologically activepeptide is linked to an immunoglobulin Fc region; and an albumin-freestabilizer.

Another object of the present invention is to provide a method forpreparing the above liquid formulation.

Still another object of the present invention is to provide acomposition for preventing or treating obesity or diabetes, comprising aliquid oxyntomodulin conjugate formulation comprising physiologicallyactive peptide oxyntomodulin linked to an immunoglobulin Fc region.

Still another object of the present invention is to provide a method forpreventing or treating obesity or diabetes, comprising administering theabove liquid formulation to a subject.

Technical Solution

To achieve the above objects, in one aspect, the present inventionprovides a liquid formulation of a long-lasting oxyntomodulin conjugate,comprising a pharmacologically effective amount of a long-lastingoxyntomodulin conjugate wherein an oxyntomodulin which is aphysiologically active peptide is linked to an immunoglobulin Fc region;and an albumin-free stabilizer, wherein the stabilizer contains abuffer, a sugar alcohol and a nonionic surfactant.

As used herein, the term “liquid formulation” refers to a drugformulation processed into a liquid form and is intended to include allliquid formulations for internal use and formulations for external use.In the prior art, the inventive liquid formulation suitable for apharmacologically effective amount of the oxyntomodulin conjugatecomprising oxyntomodulin linked to the immunoglobulin Fc domain was notreported. Thus, the liquid formulation of the present invention maycomprise a pharmacologically effective amount of the oxyntomodulinconjugate comprising oxyntomodulin linked to the immunoglobulin Fcdomain, and an albumin-free stabilizer, wherein the stabilizer containsa buffer, a sugar alcohol and a nonionic surfactant. In addition, theliquid formulation of the present invention may further comprise apreservative.

In the present invention, the stabilizer may further comprise one ormore components selected from the group consisting of isotonic agents,sugars, polyhydric alcohols, and amino acids. The sugar alcohol may beone or more selected from the group consisting of mannitol, sorbitol andglycerol, and the concentration of the sugar alcohol in the liquidformulation may be 2-15% (w/v). Further, the buffer may be one or moreselected from the group consisting of citrate, acetate, histidine andphosphate buffers and may have a pH ranging from 4.5 to 7.0. Theisotonic agent may be sodium chloride, and the nonionic surfactant maybe polysorbate or poloxamer and be present at a concentration of0.001-0.1% (w/v). The amino acid may be methionine. Thus, the liquidformulation of the present invention may comprise a stabilizer thatcontains a buffer having a pH ranging from 4.8 to 6.0, one or more sugaralcohols selected from the group consisting of mannitol and sorbitol,and polysorbate 20.

In addition, the liquid formulation of the present invention may furthercomprise one or more preservatives selected from the group consisting ofm-cresol, phenol and benzyl alcohol. The concentration of thepreservative in the liquid formulation may be 0.001-1% (w/v).

Particularly, the liquid formulation of the present invention maycomprise a pharmacologically effective amount of the long-lastingoxyntomodulin conjugate, 5-50 mM histidine, 2-15% (w/v) of mannitol,0.01-1 mg/mL of methionine and 0.001-0.1% (w/v) of polysorbate 20. Inaddition to these components, the liquid formulation may furthercomprise 0.001-1% (w/v) of m-cresol.

As used herein, the term “stabilizer” refers to a substance that stablymaintains ingredients such as active ingredients for a specific periodof time. For the purpose of the present invention, the term refers to asubstance that enables the long-lasting oxyntomodulin conjugate to bestably stored. The storage stability of proteins such as thelong-lasting oxyntomodulin conjugate is important not only to guaranteea precise dose, but also to inhibit the potential production of anantigenic substance for the oxyntomodulin derivative conjugate.

The stabilizer in the present invention preferably contains a buffer, asugar alcohol and a nonionic surfactant in order to impart stability tothe long-lasting oxyntomodulin conjugate. In addition, the stabilizermay preferably further comprise one or more components selected from thegroup consisting of isotonic agents, sugars, polyhydric alcohols andamino acids.

The buffer functions to maintain the pH of the liquid formulation sothat the pH of the liquid formulation does not rapidly change so as tomake the long-lasting oxyntomodulin conjugate stable. Examples of thebuffer may include pharmaceutically acceptable pH buffers, including analkali salt (sodium phosphate, potassium phosphate, or a hydrogen ordihydrogen salt thereof), sodium citrate, citric acid, sodium acetate,acetic acid, and histidine, or a mixture of these buffers may also beused. The buffer is preferably a citrate or histidine buffer, and morepreferably a histidine buffer. The concentration of the buffer ispreferably 5-100 mM, and more preferably 5-50 mM. The pH of the bufferis preferably 4.0-8.0, more preferably 4.5-7.0, and even more preferably5.0-6.0.

In an example of the present invention, the stability of thelong-lasting oxyntomodulin conjugate according to the pH of the bufferof the liquid formulation was measured. That is, after the long-lastingoxyntomodulin conjugate was stored at 25° C. for 0-4 weeks whilechanging the pH of the buffer, the remaining amount of the conjugate wasanalyzed, and as a result, it was shown that the oxyntomodulin conjugatewas more stable at pH 5.6, pH 5.8 and pH 6.0 (Example 3, Tables 2 to 5,Example 7, Tables 18 to 21, Example 8 and Tables 22 to 25). Thus, it wasfound that the pH of the most stable buffer in the present inventionranges from 5.0 to 6.0. In an example of the present invention, thestability of the long-lasting oxyntomodulin conjugate according to thekind of buffer of the liquid formulation was measured. Specifically,after the oxyntomodulin conjugate was stored with 0.02% polysorbate 20,0.1 mg/mL of methionine and 5% mannitol at 25° C. for 0-4 weeks, theremaining amount of the conjugate was analyzed. The results of SE-HPLCanalysis indicated that the remaining amount of the conjugate did notgreatly differ between the buffers at the same pH. The results ofIE-HPLC or RP-HPLC analysis indicated that histidine was most stable atthe same pH (Example 8 and Tables 22 to 25).

The sugar alcohol functions to increase the stability of thelong-lasting oxyntomodulin conjugate. In the present invention, thesugar alcohol may be one or more selected from the group consisting ofmannitol, sorbitol and glycerol. Preferably, the sugar alcohol may bemannitol. The concentration of the sugar alcohol in the liquidformulation is preferably 1-20% (w/v), and more preferably 2-15% (w/v).

In an example of the present invention, the influence of the kind ofsugar alcohol as a stabilizer on the stability of the long-lastingoxyntomodulin conjugate was analyzed. Specifically, the oxyntomodulinconjugate was stored in citrate buffer (pH 5.6) at 25° C. for 0-4 weeks,and then analyzed by IE-HPLC, SE-HPLC and RP-HPLC. As a result, theconjugate was more stable in the presence of mannitol or sorbitol thanin the presence of glycerol at the same concentration. The results ofRP-HPLC analysis indicated that the conjugate was a little more stablein the presence of mannitol compared to the presence of sorbitol(Example 4 and Tables 6 to 9). In other words, it was shown that theaddition of mannitol or sorbitol showed excellent stability, but theconjugate was most stable in the presence of mannitol.

In an example of the present invention, the influence of theconcentration of the sugar alcohol as a stabilizer on the stability ofthe long-lasting oxyntomodulin conjugate was analyzed. Specifically, theoxyntomodulin conjugate was stored at 25° C. for 0-4 weeks, and thenanalyzed by IE-HPLC, SE-HPLC and RP-HPLC. As a result, in the presenceof 2% mannitol or 15% mannitol, a protein precipitate was produced, andin the presence of 5% mannitol or 10% mannitol, the conjugate was stable(Example 5, Tables 10 to 13, Example 7 and Tables 18 to 21).

The nonionic surfactant functions to lower the surface tension of theprotein solution to prevent the protein from being adsorbed onto ahydrophobic surface or from aggregating. Preferred examples of anonionic surfactant that may be used in the present invention includepolysorbate-based nonionic surfactants and poloxamer-based nonionicsurfactants, which may be used alone or in combination of two or more.It is not proper that the nonionic surfactant is used at highconcentrations in the liquid formulation. The liquid formulation of thepresent invention contains the nonionic surfactant at a concentration of0.2% (w/v) or less, and preferably 0.001-0.1% (w/v).

The stabilizer of the present invention may contain an amino acid suchas methionine. Methionine functions to additionally stabilize theprotein by inhibiting the production of impurities that can be causedby, for example, the oxidative reaction of the protein.

In an example of the present invention, the influence of theconcentration of the nonionic surfactant as a stabilizer and thepresence or absence of an amino acid on the stability of thelong-lasting oxyntomodulin conjugate was tested. Specifically, theoxyntomodulin conjugate was stored in citrate buffer (pH 5.6) and 10%mannitol at 25° C. for 0-4 weeks, and then analyzed by IE-HPLC, SE-HPLCand RP-HPLC. As a result, the oxyntomodulin conjugate was most stable inthe presence of 0.02% polysorbate 20 and 0.1 mg/mL of methionine(Example 6 and Tables 14 to 17).

The isotonic agent functions to maintain osmotic pressure a suitablelevel when administering the long-lasting oxyntomodulin conjugate insolution in vivo and may additionally function to further stabilize thelong-lasting oxyntomodulin conjugate in solution. Typical examples ofthe isotonic agent include water-soluble inorganic salts, such as sodiumchloride, sodium sulfate, sodium citrate and the like. The concentrationof the isotonic agent is preferably 0-200 mM, and the content thereofcan be suitably controlled.

The stabilizer of the present invention preferably contains no albumin.Human serum albumin that can be used as a protein stabilizer is preparedfrom human blood, and thus can be contaminated with human pathogenicvirus, and gelatin or bovine serum albumin can cause diseases or cancause allergic reactions in some patients. The albumin-free stabilizerof the present invention does not contain a foreign protein such ashuman or animal serum albumin or purified gelatin, and thus is notsusceptible to viral infection.

Preferred examples of sugars among the sugars and polyhydric alcoholsthat may additionally be used to increase the storage stability of thelong-lasting oxyntomodulin conjugate include monosaccharides such asmannose, glucose, fucose and xylose, and polysaccharides such aslactose, maltose, sucrose, raffinose and dextran, and preferred examplesof the polyhydric alcohols include polypropylene, low-molecular-weightpolyethylene glycol, glycerol, low-molecular-weight polypropylene glycoland the like. These sugars and polyhydric alcohols may be used alone orin combination of two or more.

In addition to the above-described buffer, isotonic agent, sugaralcohol, amino acid and nonionic surfactant, the liquid formulation ofthe present invention may further comprise other components orsubstances known in the art within a range that does not impair theeffect of the present invention.

The inventive liquid formulation of a long-lasting oxyntomodulinconjugate comprises a pharmacologically effective amount of thelong-lasting oxyntomodulin conjugate comprising physiologically activepeptide oxyntomodulin linked to a immunoglobulin Fc region, and analbumin-free stabilizer, wherein the stabilizer may contain a bufferhaving a pH ranging from 4.8 to 7.0, one or more sugar alcohol selectedfrom the group consisting of mannitol and sorbitol, and polysorbate 20.More specifically, the stabilizer may contain a buffer having a pHranging from 5.0 to 6.0, mannitol and polysorbate 20. In addition, thestabilizer may further comprise one or more components selected from thegroup consisting of isotonic agents, sugars, polyhydric alcohols andamino acids.

The inventive albumin-free liquid formulation containing a highconcentration of the long-lasting oxyntomodulin conjugate, which impartsstability to the long-lasting oxyntomodulin conjugate, is notsusceptible to viral infection, is simple and shows excellent storagestability, and thus can be provided in an economical manner compared toother stabilizers or lyophilized formulations.

In addition, because the liquid formulation of the present inventioncomprises the long-lasting oxyntomodulin conjugate that hasphysiological activity for an extended period of time compared to nativeoxyntomodulin, it can maintain protein activity in the human body for anextended period of time compared to conventional oxyntomodulinformulations, and thus can be used as an efficient drug formulation. Inaddition, the liquid formulation of the present invention impartsexcellent stability even to a high concentration of the long-lastingoxyntomodulin conjugate.

As used herein, the term “oxyntomodulin” refers to a peptide producedfrom pre-glucagon that is a precursor of glucagon. In the presentinvention, oxyntomodulin is meant to include native oxyntomodulin andits precursor, derivative, fragment and variant. Preferably,oxyntomodulin has an amino acid sequence of SEQ ID NO: 1(HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA).

As used herein, the term “oxyntomodulin derivative” is meant to includea peptide, a peptide derivative or a peptide mimic that is obtained bythe addition, deletion or substitution of amino acids in the amino acidsequence of oxyntomodulin and can activate the glucagon and GLP-1receptors at a higher level than that activated by native oxyntomodulin.In the present invention, the oxyntomodulin derivative may have any oneof amino acid sequences of SEQ ID NOS: 2 to 34. Preferably, theoxyntomodulin derivative may have an amino acid sequence of SEQ ID NO:23 or 25. More preferably, it may have an amino acid sequence of SEQ IDNO: 25.

As used herein, the term “oxyntomodulin fragment” refers to a fragmenthaving one or more amino acids at the amino or carboxyl terminal end ofnative oxyntomodulin, in which the added amino acids may also benon-naturally occurring amino acids (e.g., D-type amino acid). Thisoxyntomodulin fragment has a function of regulating blood glucose levelsin vivo.

As used herein, the term “oxyntomodulin variant” is a peptide that hasone or more amino acid residues different from those of the amino acidsequence of native oxyntomodulin and possesses a function of activatingthe GLP-1 and glucagon receptors. The oxyntomodulin variant can beprepared by any one of substitution, addition, deletion, modification,or a combination thereof of some amino acids in the amino acid sequenceof native oxyntomodulin.

Methods for preparing the oxyntomodulin variant, derivative and fragmentmay be used alone or in combination. For example, the present inventionalso includes a peptide, which has one or more amino acids differentfrom those of native oxyntomodulin and deamination of the N-terminalamino acid residues and has a function of activating both GLP-1 receptorand glucagon receptor.

Amino acids mentioned herein are abbreviated according to thenomenclature rules of IUPAC-IUB as follows:

Alanine A;

Arginine R;

Asparagine N;

Aspartic acid D;

Cysteine C;

Glutamic acid E;

Glutamine Q;

Glycine G;

Histidine H;

Isoleucine I;

Leucine L;

Lysine K;

Methionine M;

Phenylalanine F

Proline P;

Serine S;

Threonine T;

Tryptophan W;

Tyrosine Y;

Valine V.

In the present invention, the oxyntomodulin derivative encompasses anypeptide, which is prepared by the substitution, addition, deletion orpost-translational modification (e.g., methylation, acylation,ubiquitination, or intramolecular covalent bonding) of amino acids inthe amino acid sequence of SEQ ID NO: 1 and can activate both theglucagon and GLP-1 receptors. For substitution or addition of the aminoacids, not only 20 amino acids commonly found in human proteins, butalso atypical or non-naturally occurring amino acids can be used.Commercial sources of atypical amino acids include Sigma-Aldrich,ChemPep Inc., and Genzyme Pharmaceuticals. The peptides, which includethese amino acids, and atypical peptide sequences may be synthesized andpurchased from commercial suppliers, for example, American PeptideCompany or Bachem (USA) or Anygen (Korea).

In a specific embodiment of the present invention, the oxyntomodulinderivative of the present invention is a novel peptide including theamino acid sequence of the following formula 1:

R1-X1-X2-GTFTSD-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X19-X20-X21-X22-X23-X24-R2  Formula1

whereinR1 is histidine, desamino-histidyl, dimethyl-histidyl(N-dimethyl-histidyl), beta-hydroxyimidazopropionyl, 4-imidazoacetyl,beta-carboxy imidazopropionyl or tyrosine;X1 is Aib (aminosiobutyric acid), d-alanine, glycine, Sar(N-methylglycine), serine or d-serine;X2 is glutamic acid or glutamine;X3 is leucine or tyrosine;X4 is serine or alanine;X5 is lysine or arginine;X6 is glutamine or tyrosine;X7 is leucine or methionine;X8 is aspartic acid or glutamic acid;X9 is glutamic acid, serine or alpha-methyl-glutamic acid or is deleted;X10 is glutamine, glutamic acid, lysine, arginine or serine or isdeleted;X11 is alanine, arginine or valine or is deleted;X12 is alanine, arginine, serine or valine or is deleted;X13 is lysine, glutamine, arginine or alpha-methyl-glutamic acid or isdeleted;X14 is aspartic acid, glutamic acid or leucine or is deleted;X15 is phenylalanine or is deleted;X16 is isoleucine or valine or is deleted;X17 is alanine, cysteine, glutamic acid, lysine, glutamine oralpha-methyl-glutamic acid or is deleted;X18 is tryptophan or is deleted;X19 is alanine, isoleucine, leucine, serine or valine or is deleted;X20 is alanine, lysine, methionine, glutamine or arginine or is deleted;X21 is asparagine or is deleted;X22 is alanine, glycine or threonine or is deleted;X23 is cysteine or lysine or is deleted;X24 is a peptide having 2 to 10 amino acids consisting of a combinationof alanine, glycine and serine or is deleted; andR2 is KRNRNNIA (SEQ ID NO: 35), GPSSGAPPPS (SEQ ID NO: 36), GPSSGAPPPSK(SEQ ID NO: 37), HSQGTFTSDYSKYLD (SEQ ID NO: 38), HSQGTFTSDYSRYLDK (SEQID NO: 39), HGEGTFTSDLSKQMEEEAVK (SEQ ID NO: 40) or is deleted (with theexception of the case in which the amino acid sequence of formula 1 isidentical to that of SEQ ID NO: 1).

In order to increase the activity of wild-type oxyntomodulin for theglucagon receptor and the GLP-1 receptor, the oxyntomodulin derivativeof the present invention may be substituted with 4-imidazoacetylobtained by deletion of the alpha carbon of histidine at position 1 ofthe amino acid sequence of SEQ ID NO: 1, desamino-histidyl obtained bydeletion of the N-terminal amino group, dimethyl-histidyl(N-dimethyl-histidyl) obtained by modification of the N-terminal aminogroup with two methyl groups, beta-hydroxy imidazopropionyl obtained bysubstitution of the N-terminal amino group with a hydroxyl group, orbeta-carboxy imidazopropionyl obtained by substitution of the N-terminalamino group with a carboxyl group. In addition, the GLP-1receptor-binding region may be substituted with amino acids that enhancehydrophobic and ionic bonds or a combination thereof. Further, a portionof the oxyntomodulin sequence may be substituted with the amino acidsequence of GLP-1 or Exendin-4 to increase the activity of the GLP-1receptor.

Moreover, a portion of the oxyntomodulin sequence may be substitutedwith a sequence that enhances alpha helix. Preferably, amino acids atpositions 10, 14, 16, 20, 24 and 28 of the amino acid sequence offormula 1 may be substituted with amino acids or amino acid derivativesconsisting of Tyr(4-Me), Phe, Phe(4-Me), Phe(4-Cl), Phe(4-CN),Phe(4-NO₂), Phe(4-NH₂), Phg, Pal, Nal, Ala(2-thienyl) andAla(benzothienyl) that are known to stabilize alpha helix, and the typeand number of alpha helix-stabilizing amino acid or amino acidderivatives to be inserted are not limited. Preferably, amino acids atpositions 10 and 14, 12 and 16, 16 and 20, 20 and 24, and 24 and 28 ofthe amino acid sequence may also be substituted with glutamic acid orlysine so as to form rings, and the number of rings to be inserted isnot limited. Most preferably, the oxyntomodulin derivative may have anamino acid sequence selected from among the following formulas 2 to 6.

In a specific embodiment, the oxyntomodulin derivative of the presentinvention is a novel peptide including the amino acid sequence of thefollowing formula 2, obtained by substitution of the amino acid sequenceof oxyntomodulin with that of exendin or GLP-1:

R1-A-R3  Formula 2

In another specific embodiment, the oxyntomodulin derivative of thepresent invention is a novel peptide including the amino acid sequenceof the following formula 3, which is prepared by linking a portion ofthe amino acid sequence of oxyntomodulin and a portion of the amino acidsequence of exendin or GLP-1 via a proper amino acid linker:

R1-B-C-R4  Formula 3

In still another specific embodiment, the oxyntomodulin derivative ofthe present invention is a novel peptide including the amino acidsequence of the following formula 4, wherein a portion of the amino acidsequence of oxyntomodulin is substituted with an amino acid thatenhances the hydrophobic binding to GLP-1 receptor. For example, it is apeptide wherein Leu at position 26 is substituted with the amino acidIle or Val that increases hydrophobicity.

R1-SQGTFTSDYSKYLD-D1-D2-D3-D4-D5-LFVQW-D6-D7-N-D8-R3  Formula 4

In still another specific embodiment, the oxyntomodulin derivative ofthe present invention is a novel peptide including the amino acidsequence of the following formula 5, wherein a portion of the amino acidsequence of native oxyntomodulin is deleted, added, or substituted withother amino acids in order to increase the abilities of nativeoxyntomodulin to activate GLP-1 receptor and glucagon receptor:

R1-E1-QGTFTSDYSKYLD-E2-E3-RA-E4-E5-FV-E6-WLMNT-E7-R5  Formula 5

In formulas 2 to 5, R1 is as described in formula 1;

A is selected from the group consisting of

(SEQ ID NO: 41) SQGTFTSDYSKYLDSRRAQDFVQWLMNT, (SEQ ID NO: 42)SQGTFTSDYSKYLDEEAVRLFIEWLMNT, (SEQ ID NO: 43)SQGTFTSDYSKYLDERRAQDFVAWLKNT, (SEQ ID NO: 44)GQGTFTSDYSRYLEEEAVRLFIEWLKNG, (SEQ ID NO: 45)GQGTFTSDYSRQMEEEAVRLFIEWLKNG, (SEQ ID NO: 46)GEGTFTSDLSRQMEEEAVRLFIEWAA, and (SEQ ID NO: 47)SQGTFTSDYSRQMEEEAVRLFIEWLMNG;

B is selected from the group consisting of

(SEQ ID NO: 41) SQGTFTSDYSKYLDSRRAQDFVQWLMNT, (SEQ ID NO: 42)SQGTFTSDYSKYLDEEAVRLFIEWLMNT, (SEQ ID NO: 43)SQGTFTSDYSKYLDERRAQDFVAWLKNT, (SEQ ID NO: 44)GQGTFTSDYSRYLEEEAVRLFIEWLKNG, (SEQ ID NO: 45)GQGTFTSDYSRQMEEEAVRLFIEWLKNG, (SEQ ID NO: 46)GEGTFTSDLSRQMEEEAVRLFIEWAA, (SEQ ID NO: 47)SQGTFTSDYSRQMEEEAVRLFIEWLMNG, (SEQ ID NO: 48) GEGTFTSDLSRQMEEEAVRLFIEW,and (SEQ ID NO: 49) SQGTFTSDYSRYLD;

C is a peptide having 2 to 10 amino acids consisting of a combination ofalanine, glycine and serine;

D1 is serine, glutamic acid or arginine;

D2 is arginine, glutamic acid or serine;

D3 is arginine, alanine or valine;

D4 is arginine, valine or serine;

D5 is glutamine, arginine or lysine;

D6 is isoleucine, valine or serine;

D7 is methionine, arginine or glutamine;

D8 is threonine, glycine or alanine;

E1 is serine, Aib, Sar, d-alanine or d-serine;

E2 is serine or glutamic acid;

E3 is arginine or lysine;

E4 is glutamine or lysine;

E5 is aspartic acid or glutamic acid;

E6 is glutamine, cysteine or lysine;

E7 is cysteine or lysine or is deleted;

R3 is KRNRNNIA (SEQ ID NO: 35), GPSSGAPPPS (SEQ ID NO: 36) orGPSSGAPPPSK (SEQ ID NO: 37);

R4 is HSQGTFTSDYSKYLD (SEQ ID NO: 38), HSQGTFTSDYSRYLDK (SEQ ID NO: 39)or HGEGTFTSDLSKQMEEEAVK (SEQ ID NO: 40); and,

R5 is KRNRNNIA (SEQ ID NO: 35), GPSSGAPPPS (SEQ ID NO: 36) orGPSSGAPPPSK (SEQ ID NO: 37) or is deleted (with the exception of thecase in which the amino acid sequences of formulas 2 to 5 are identicalto that of SEQ ID NO: 1).

Preferably, the oxyntomodulin derivative of the present invention may bea novel peptide of the following formula 6:

R1-X1-X2-GTFTSD-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X19-X20-X21-X22-X23-X24-R2  Formula6

wherein R1 is histidine, desamino-histidyl, 4-imidazoacetyl or tyrosine;X1 is Aib(aminosiobutyric acid), glycine or serine;X2 is glutamic acid or glutamine; X3 is leucine or tyrosine;X4 is serine or alanine;X5 is lysine or arginine;X6 is glutamine or tyrosine;X7 is leucine or methionine; X8 is aspartic acid or glutamic acid;X9 is glutamic acid or alpha-methyl-glutamic acid or is deleted;X10 is glutamine, glutamic acid, lysine or arginine or is deleted;X11 is alanine or arginine or is deleted;X12 is alanine or valine or is deleted;X13 is lysine, glutamine, arginine or alpha-methyl-glutamic acid or isdeleted;X14 is aspartic acid, glutamic acid or leucine or is deleted;X15 is phenylalanine or is deleted;X16 is isoleucine or valine or is deleted;X17 is alanine, cysteine, glutamic acid, glutamine oralpha-methyl-glutamic acid or is deleted;X18 is tryptophan or is deleted;X19 is alanine, isoleucine, leucine or valine or is deleted;X20 is alanine, lysine, methionine or arginine or is deleted;X21 is asparagine or is deleted;X22 is threonine or is deleted;X23 is cysteine, lysine or is deleted;X24 is a peptide having 2 to 10 amino acids consisting of glycine or isdeleted; andR2 is KRNRNNIA (SEQ ID NO: 35), GPSSGAPPPS (SEQ ID NO: 36), GPSSGAPPPSK(SEQ ID NO: 37), HSQGTFTSDYSKYLD (SEQ ID NO: 38), HSQGTFTSDYSRYLDK (SEQID NO: 39) or HGEGTFTSDLSKQMEEEAVK (SEQ ID NO: 40) or is deleted (withthe exception of the case in which the amino acid sequence of formula 6is identical to that of SEQ ID NO: 1).

More preferably, the oxyntomodulin derivative of the present inventionmay be selected from the group consisting of the peptides of SEQ ID NOs:2 to 34. Even more preferably, the oxyntomodulin derivative of thepresent invention may be an oxyntomodulin derivative described in Table1 of Example 1.

Oxyntomodulin has the activities of two peptides, GLP-1 and glucagon.GLP-1 has the effect of lowering blood glucose levels by insulinsecretion, but glucagon has the effect of increasing blood glucoselevels. Further, GLP-1 inhibits food intake and suppresses gastricemptying, and glucagon has the effect of reducing bodyweight byfacilitating lipolysis and increasing energy metabolisms. Thus, GLP-1and glucagon have different biological effects. Thus, in the case inwhich the two peptides present as a conjugate, if the effect of any oneof the two peptides is greater than that of the other, an adverse effectcan occur. For example, if the effect of glucagon is greater than thatof GLP-1, blood glucose levels can rise, and if the effect of GLP-1 isgreater than that of glucagon, side effects such as nausea and vomitingcan occur. In addition, the effect of the two peptides may varydepending on the ratio of the activities of the two peptides. Thus, theoxyntomodulin derivatives and their conjugates are not limited only toderivatives having increased activities.

As used herein, the term “oxyntomodulin conjugate” refers to a conjugatecomprising oxyntomodulin and another element. The other element may beany substance having beneficial functions, including increasing theblood half-life of oxyntomodulin or delaying the release ofoxyntomodulin into the kidneys. The conjugate of the present inventioncan bind covalently to oxyntomodulin or form microspheres to increasethe serum stability of oxyntomodulin or to delay the release ofoxyntomodulin into the kidneys or to change the binding activity ofoxyntomodulin to its receptor. The carrier that can form a conjugatecomprising oxyntomodulin may be selected from the group consisting ofalbumin, transferrin, antibodies, antibody fragments, elastin, heparin,polysaccharide such as chitin, fibronectin, and the like, which can bindto oxyntomodulin to increase the serum stability of oxyntomodulin.Preferably, the carrier is an immunoglobulin Fc region.

The immunoglobulin Fc that may be used in the present invention may be ahuman immunoglobulin Fc, an immunoglobulin Fc having the sequence of ananalogue thereof, or an immunoglobulin Fc derived from animals,including cows, goats, pigs, mice, rabbits, hamsters, rats and guineapigs. Further, the immunoglobulin Fc region may be derived from IgG,IgA, IgD, IgE, IgM, or a combination or hybrid thereof. Further, eachdomain of the immunoglobulin Fc region of the present invention may be ahybrid of domains originated from different immunoglobulins selectedfrom the group consisting of IgG, IgA, IgD, IgE, and IgM. Alternatively,the immunoglobulin Fc region is a dimer or multimer consisting ofsingle-chain immunoglobulins composed of domains of the sane origin.Preferably, the immunoglobulin Fc region is one derived from IgG or IgM,which is most rich in human blood. Most preferably, it is animmunoglobulin Fc derived from IgG known to increase the half-life ofligand-binding proteins. The immunoglobulin Fc may be prepared either bytreating native IgG with a specific protease or from transformed cellsusing recombination technique. Preferably, it is a recombinant humanimmunoglobulin Fc prepared in E. coli.

Meanwhile, IgG can also be sub-classified into IgG1, IgG2, IgG3 andIgG4, and in the present invention, a combination or hybrid of thesesubclasses is also possible. Preferably, IgG is the IgG2 ad IgG4subclass, and most preferably, it is the Fc region of IgG4 thatsubstantially lacks effector functions such as complement-dependentcytotoxicity (CDC). In other words, the most preferred immunoglobulin Fcregion that is used as a drug carrier in the present invention is anon-glycosylated Fc region derived from human IgG4. A human-derived Fcregion is more preferable than a non-human-derived Fc region, which mayact as an antigen in the human body and cause undesirable immuneresponses such as the production of a new antibody against the antigen.

In the present invention, the oxyntomodulin conjugate may be prepared byusing a non-peptidyl polymer or by gene recombination technique.Preferably, the conjugate may be prepared by linking oxyntomodulin tothe immunoglobulin Fc region by a non-peptidyl polymer.

The non-peptidyl polymer may be linked to each of oxyntomodulin and theimmunoglobulin Fc region. Each end of the non-peptidyl polymer may belinked to the immunoglobulin Fc region and the amine or thiol group ofthe oxyntomodulin derivative, respectively.

As used herein, the term “oxyntomodulin conjugate” refers to one havingan increased long-lasting effect compared to native oxyntomodulin.Examples of the long-lasting conjugate include, but are not limited to,a conjugate in which a oxyntomodulin derivative resulting from themodification, substitution, addition or deletion of amino acids in theamino acid sequence of native oxyntomodulin is linked to a biodegradablepolymer such as polyethylene glycol (PEG), a conjugate in which aprotein having excellent long-lasting properties, such as albumin orimmunoglobulin, is linked to oxyntomodulin, a conjugate in which a fattyacid having ability to bind to albumin in vivo is linked tooxyntomodulin, or a conjugate in which oxyntomodulin is encapsulated inbiodegradable nanoparticles.

As used herein, the term “non-peptidyl polymer” refers to abiocompatible polymer including two or more repeating units linked toeach other by any covalent bond in place of a peptide bond. In thepresent invention, the non-peptidyl polymer may be used interchangeablywith the non-peptidyl linker.

A peptide linker that is used in a fusion protein obtained by aconventional inframe fusion method has drawbacks in that it is easilycleaved by proteinase in vivo, and thus the desired effect of increasingthe serum half-life of the active drug by a carrier cannot be obtained.However, in the present invention, the polymer having resistance toproteinase can be used to maintain the serum half-life of the peptide,similar to the carrier. Therefore, any non-peptidyl polymer can be usedwithout limitation in the present invention, as long as it is a polymerhaving the aforementioned function, that is, a polymer having resistanceto proteinase in vivo. The non-peptidyl polymer has a molecular weightin the range of 1 to 100 kDa, and preferably 1 to 20 kDa. Thenon-peptidyl polymer of the present invention, which is linked to theimmunoglobulin Fc region, may be one kind of polymer or a combination ofdifferent polymers.

The non-peptidyl polymer that is used in the present invention may havea reactive group capable of binding to the immunoglobulin Fc region andthe protein drug. The reactive group at both ends of the non-peptidylpolymer is preferably selected from the group consisting of a reactivealdehyde group, a propionaldehyde group, a butyraldehyde group, amaleimide group and a succinimide derivative.

The succinimide derivative may be succinimidyl propionate, hydroxysuccinimidyl, succinimidyl carboxymethyl, or succinimidyl carbonate. Inparticular, when the non-peptidyl polymer has a reactive aldehyde groupat both ends thereof, non-specific reactions can be minimized, and aphysiologically active polypeptide and an immunoglobulin can beeffectively bound to both ends of the non-peptidyl polymer,respectively. A final product generated by reductive alkylation with analdehyde bond is much more stable than that linked by an amide bond. Thealdehyde reactive group selectively binds to an N-terminus at a low pHand can form a covalent bond with a lysine residue at a high pH such aspH 9.0.

The reactive groups at both ends of the non-peptidyl polymer may be thesame or different. For example, the non-peptidyl polymer may possess amaleimide group at one end, and an aldehyde group, a propionaldehydegroup or a butyraldehyde group at the other end. When a polyethyleneglycol having a reactive hydroxy group at both ends thereof is used asthe non-peptidyl polymer, the hydroxy group may be activated to variousreactive groups by known chemical reactions, or a polyethylene glycolhaving a commercially available modified reactive group may be used soas to prepare the long acting conjugate of the present invention.

The conjugate of the present invention may be one in which each end ofthe non-peptidyl polymer is linked to the immunoglobulin Fc region andthe amine or thiol group of the oxyntomodulin derivative, respectively.

Meanwhile, in the present invention, both ends of the non-peptidylpolymer include reactive groups to which an immunoglobulin Fc region anda protein drug can bind. Examples of the reactive groups include, butare not limited to, an aldehyde group, a propionaldehyde group or abutyraldehyde group, a maleimide group, a succinimide derivative(succinimidyl propionate, hydroxyl succinimidyl, succinimidylcarboxymethyl or succinimidyl carbonate) and the like.

The reactive groups at both ends of the linker that is the non-peptidylpolymer may be the same or different. For example, the non-peptidylpolymer may have a maleimide group at one end and an aldehyde group, apropionaldehyde group or a butyraldehyde group at the other end. Forexample, when the non-peptidyl polymer has a reactive aldehyde group atone end and a reactive maleimide group at the other end, non-specificreactions can be minimized, and a physiologically active polypeptide andan immunoglobulin can be effectively bound to both ends of thenon-peptidyl polymer. The non-peptidyl polymer that may be used in thepresent invention may be selected from the group consisting ofpolyethylene glycol, polypropylene glycol, an ethylene glycol/propyleneglycol copolymer, polyoxyethylated polyol, polyvinyl alcohol,polysaccharides, dextran, polyvinyl ethyl ether, biodegradable polymerssuch as PLA (poly(lactic acid)) and PLGA (polylactic-glycolic acid),lipid polymers, chitins, hyaluronic acid, and combinations thereof.Preferably, the non-peptidyl polymer is polyethylene glycol. Inaddition, derivatives thereof known in the art and derivatives that maybe easily prepared by a method known in the art are included in scope ofthe present invention.

In an example of the present invention, a conjugate was synthesized bylinking oxyntomodulin or its derivative to the immunoglobulin Fc regionvia a covalent bond using the non-peptidyl polymer PEG including apropionaldehyde group alone or both a maleimide group and an aldehydegroup.

The conjugate of the present invention has excellent GLP-1 receptor andglucagon receptor activities compared to native oxyntomodulin. Also, ithas bound thereto an Fc region that increase the in vivo blood half-lifethereof to maintain the activity thereof in vivo for an extended periodof time.

As used herein, term “preservative” refers to a substance that is usedto prevent abnormal reactions or decomposition from being caused bymicrobial contamination. The liquid formulation of the present inventionmay further comprise a preservative. A preservative is generally used inmultiple-dosage formulations that are most likely contaminated withmicroorganisms, but is not limited thereto and may also be used inlyophilized formulations or single-dosage formulations to preventmicrobial contamination. The liquid formulation of the present inventionmay comprise one or more preservatives selected from m-cresol, phenoland benzyl alcohol. The concentration of the preservative in the liquidformulation may be 0.001-1% (w/v). Particularly, the preservative thatis included in the liquid formulation of the present invention ispreferably m-cresol. The liquid formulation of the present invention maybe a multiple-dosage formulation.

In another aspect, the present invention provides a method for preparinga liquid formulation of a long-lasting oxyntomodulin conjugate.

Specifically, in one embodiment of the present invention, the method forpreparing the liquid formulation may comprise the steps of: a) preparinga long-lasting oxyntomodulin conjugate; and b) mixing the preparedlong-lasting oxyntomodulin conjugate with a stabilizer containing abuffer, a sugar alcohol and a non-ionic surfactant.

In another embodiment of the present invention, the method for preparingthe liquid formulation may comprise the steps of: a) preparing along-lasting oxyntomodulin conjugate; and b) mixing the preparedlong-lasting oxyntomodulin conjugate with a stabilizer, which contains abuffer, a sugar alcohol and a non-ionic surfactant, and a preservative.

Preferably, the stabilizer in step b) may further comprise one or moreselected from the group consisting of isotonic agents, sugars,polyhydric alcohols and amino acids.

In still another aspect, the present invention provides a pharmaceuticalcomposition for preventing or treating obesity or diabetes, comprisingthe above liquid formulation.

As used herein, the term “preventing” refers to all actions that inhibitor delay the development of a target disease. As used herein, the term“preventing” means administering the conjugate of the present inventionto inhibit or delay the development of diabetic conditions, such asabnormal blood glucose levels or abnormal insulation secretion, orobesity conditions such as an increase in body weight or body fat.

As used herein, the term “treating” refers to all actions thatalleviate, ameliorate or relieve the symptoms of the disease developed.As used herein, the term “treating” means administering the conjugate ofthe present invention to alleviate, ameliorate or relieve the abovediabetic conditions or obesity conditions to normalize blood glucoselevels and insulin secretion and reduce body weight or body fat.

As used herein, the term “obesity” refers to an excessive amount of bodyfat. A body mass index (=weight (kg) divided by height (m)) of 25 ormore is defined as obesity. Obesity generally results from an energyimbalance in which energy intake exceeds energy expenditure. Obesity isa metabolic disease that affects the entire body and highly likely tolead to diabetes and hyperlipidemia. In addition, obesity is related tosexual dysfunction, arthritis, and an increased risk of the developmentof cardiovascular diseases, and is also related to the development ofcancer in some cases.

As used herein the term “diabetes” is a kind of metabolic disease inwhich insulin secretion is insufficient or normal functions are notmade. Diabetes is characterized by increased blood glucose levels andcauses various health problems. In the case of diabetes, glucose isexcreted with urine.

The pharmaceutical composition of the present invention may furthercomprise a pharmaceutically acceptable carrier, excipient or diluent. Asused herein, the term “pharmaceutically acceptable” means an amount thatis sufficient to exhibit therapeutic effects and causes no side effects.The dose of the active ingredient of the pharmaceutical composition ofthe present invention may be readily determined by those skilled in theart depending on the type of disease, the patient's age, weight, healthcondition, sex, and drug sensitivity, the route of administration, themode of administration, the frequency of administration, the duration oftreatment, drugs used in combination or coincident with the compositionof this invention, and other factors known in the medical field.

In still another aspect, the present invention provides a method forpreventing or treating obesity or diabetes, comprising administering theliquid formulation to a subject.

Herein, the liquid formulation, obesity and diabetes are as describedabove.

As used herein, the term “subject” refers to a subject suspected ofhaving obesity or diabetes. Specifically, the term means mammals,including humans, rats and domestic animals, which have or are at therisk of developing the above disease. In addition, the subject may beany subject that can be treated by the liquid formulation derivative ofthe present invention.

The therapeutic method of the present invention may compriseadministering a pharmaceutically effective amount of the pharmaceuticalcomposition comprising the liquid formulation. The total daily dose ofthe composition can be determined through appropriate medical judgmentby a physician, and the composition may be administered once or severaltimes. However, in view of the purpose of the present invention, thespecific therapeutically effective dose of the composition for anyparticular patient may vary depending on various factors well known inthe medical field, including the kind and degree of response to beachieved, concrete compositions according to whether other agents areused therewith or not, the patient's age, body weight, health condition,sex and diet, the time and route of administration, the secretion rateof the composition, the duration of treatment, other drugs used incombination or coincident with the composition of the present invention,and other factors known in the medical field.

Advantageous Effects

The inventive liquid formulation comprising the long-lastingoxyntomodulin conjugate comprises a buffer, a sugar alcohol and anonionic surfactant and does not contain a human serum albumin andfactors that are potentially harmful to the human body, and thus is notsusceptible to viral infection. In addition, the oxyntomodulin conjugateof the present invention comprises oxyntomodulin linked to animmunoglobulin Fc region, and thus has a great molecular weight,prolonged physiological activity, and excellent storage stability,compared to native oxyntomodulin.

DESCRIPTION OF DRAWINGS

FIG. 1 a is a graph showing the results obtained by purifying amono-PEGylated oxyntomodulin derivative (SEQ ID NO: 25) through a SOURCES purification column.

FIG. 1 b is a graph showing the results obtained by purifying aconjugate of a mono-PEGylated oxyntomodulin derivative (SEQ ID NO: 25)and an immunoglobulin Fc through a SOURCE 15Q purification column.

FIG. 1 c is a graph showing the results obtained by purifying aconjugate of a mono-PEGylated oxyntomodulin derivative (SEQ ID NO: 25)and an immunoglobulin Fc through a SOURCE ISO purification column.

FIG. 2 a is a graphic diagram showing the results obtained by evaluatingthe stability of a long-lasting oxyntomodulin conjugate according to pHby IE-HPLC in Example 3 after 0-2 weeks of storage at 25° C. Each graphin FIG. 2 a shows the percent retention of the long-lastingoxyntomodulin conjugate relative to the start value.

FIG. 2 b is a graphic diagram showing the results obtained by evaluatingthe stability of a long-lasting oxyntomodulin conjugate according to pHby SE-HPLC in Example 3 after 0-2 weeks of storage at 25° C. Each graphin FIG. 2 b shows the percent retention of the long-lastingoxyntomodulin conjugate relative to the start value.

FIG. 2 c is a graphic diagram showing the results obtained by evaluatingthe stability of a long-lasting oxyntomodulin conjugate according to pHby RP-HPLC in Example 3 after 0-2 weeks of storage at 25° C. Each graphin FIG. 2 c shows the percent retention of the long-lastingoxyntomodulin conjugate relative to the start value.

FIG. 3 a is a graphic diagram showing the results obtained by evaluatingthe stability of a long-lasting oxyntomodulin conjugate according to thekind of sugar alcohol and the presence or absence of an isotonic agentby IE-HPLC in Example 4 after 0-4 weeks of storage at 25° C. Each graphin FIG. 3 a shows the percent retention of the long-lastingoxyntomodulin conjugate relative to the start value.

FIG. 3 b is a graphic diagram showing the results obtained by evaluatingthe stability of a long-lasting oxyntomodulin conjugate according to thekind of sugar alcohol and the presence or absence of an isotonic agentby SE-HPLC in Example 4 after 0-4 weeks of storage at 25° C. Each graphin FIG. 3 b shows the percent retention of the long-lastingoxyntomodulin conjugate relative to the start value.

FIG. 3 c is a graphic diagram showing the results obtained by evaluatingthe stability of a long-lasting oxyntomodulin conjugate according to thekind of sugar alcohol and the presence or absence of an isotonic agentby RP-HPLC in Example 4 after 0-4 weeks of storage at 25° C. Each graphin FIG. 3 c shows the percent retention of the long-lastingoxyntomodulin conjugate relative to the start value.

FIG. 4 a is a graphic diagram showing the results obtained by evaluatingthe stability of a long-lasting oxyntomodulin conjugate according to theconcentration of sugar alcohol by IE-HPLC in Example 5 after 0-4 weeksof storage at 25° C. Each graph in FIG. 4 a shows the percent retentionof the long-lasting oxyntomodulin conjugate relative to the start value.

FIG. 4 b is a graphic diagram showing the results obtained by evaluatingthe stability of a long-lasting oxyntomodulin conjugate according to theconcentration of sugar alcohol by SE-HPLC in Example 5 after 0-4 weeksof storage at 25° C. Each graph in FIG. 4 b shows the percent retentionof the long-lasting oxyntomodulin conjugate relative to the start value.

FIG. 4 c is a graphic diagram showing the results obtained by evaluatingthe stability of a long-lasting oxyntomodulin conjugate according toconcentration of sugar alcohol by RP-HPLC in Example 5 after 0-4 weeksof storage at 25° C. Each graph in FIG. 4 c shows the percent retentionof the long-lasting oxyntomodulin conjugate relative to the start value.

FIG. 5 a is a graphic diagram showing the results obtained by evaluatingthe stability of a long-lasting oxyntomodulin conjugate according to theconcentration of a surfactant and the presence or absence of an aminoacid by IE-HPLC in Example 6 after 0-4 weeks of storage at 25° C. Eachgraph in FIG. 5 a shows the percent retention of the long-lastingoxyntomodulin conjugate relative to the start value.

FIG. 5 b is a graphic diagram showing the results obtained by evaluatingthe stability of a long-lasting oxyntomodulin conjugate according to theconcentration of a surfactant and the presence or absence of an aminoacid by SE-HPLC in Example 6 after 0-4 weeks of storage at 25° C. Eachgraph in FIG. 5 b shows the percent retention of the long-lastingoxyntomodulin conjugate relative to the start value.

FIG. 5 c is a graphic diagram showing the results obtained by evaluatingthe stability of a long-lasting oxyntomodulin conjugate according to theconcentration of a surfactant and the presence or absence of an aminoacid by RP-HPLC in Example 6 after 0-4 weeks of storage at 25° C. Eachgraph in FIG. 5 c shows the percent retention of the long-lastingoxyntomodulin conjugate relative to the start value.

FIG. 6 a is a graphic diagram showing the results obtained by evaluatingthe stability of a long-lasting oxyntomodulin conjugate according to pHand the concentration of sugar alcohol by IE-HPLC in Example 7 after 0-4weeks of storage at 25° C. Each graph in FIG. 6 a shows the percentretention of the long-lasting oxyntomodulin conjugate relative to thestart value.

FIG. 6 b is a graphic diagram showing the results obtained by evaluatingthe stability of a long-lasting oxyntomodulin conjugate according to pHand the concentration of sugar alcohol by SE-HPLC in Example 7 after 0-4weeks of storage at 25° C. Each graph in FIG. 6 b shows the percentretention of the long-lasting oxyntomodulin conjugate relative to thestart value.

FIG. 6 c is a graphic diagram showing the results obtained by evaluatingthe stability of a long-lasting oxyntomodulin conjugate according to pHand the concentration of sugar alcohol by RP-HPLC in Example 7 after 0-4weeks of storage at 25° C. Each graph in FIG. 6 c shows the percentretention of the long-lasting oxyntomodulin conjugate relative to thestart value.

FIG. 7 a is a graphic diagram showing the results obtained by evaluatingthe stability of a long-lasting oxyntomodulin conjugate according to pHand the kind of buffer alcohol by IE-HPLC in Example 8 after 0-4 weeksof storage at 25° C. Each graph in FIG. 7 a shows the percent retentionof the long-lasting oxyntomodulin conjugate relative to the start value.

FIG. 7 b is a graphic diagram showing the results obtained by evaluatingthe stability of a long-lasting oxyntomodulin conjugate according to pHand the kind of buffer alcohol by SE-HPLC in Example 8 after 0-4 weeksof storage at 25° C. Each graph in FIG. 7 b shows the percent retentionof the long-lasting oxyntomodulin conjugate relative to the start value.

FIG. 7 c is a graphic diagram showing the results obtained by evaluatingthe stability of a long-lasting oxyntomodulin conjugate according to pHand the kind of buffer alcohol by RP-HPLC in Example 8 after 0-4 weeksof storage at 25° C. Each graph in FIG. 7 c shows the percent retentionof the long-lasting oxyntomodulin conjugate relative to the start value.

FIG. 8 a is a graphic diagram showing the results obtained by evaluatingthe stability of a long-lasting oxyntomodulin conjugate according to thepresence or absence of a preservative and the concentration oflong-lasting oxyntomodulin by IE-HPLC in Example 9 after 0-4 weeks ofstorage at 25° C. Each graph in FIG. 8 a shows the percent retention ofthe long-lasting oxyntomodulin conjugate relative to the start value.

FIG. 8 b is a graphic diagram showing the results obtained by evaluatingthe stability of a long-lasting oxyntomodulin conjugate according to thepresence or absence of a preservative and the concentration oflong-lasting oxyntomodulin by SE-HPLC in Example 9 after 0-4 weeks ofstorage at 25° C. Each graph in FIG. 8 b shows the percent retention ofthe long-lasting oxyntomodulin conjugate relative to the start value.

FIG. 8 c is a graphic diagram showing the results obtained by evaluatingthe stability of a long-lasting oxyntomodulin conjugate according to thepresence or absence of a preservative and the concentration oflong-lasting oxyntomodulin by RP-HPLC in Example 9 after 0-4 weeks ofstorage at 25° C. Each graph in FIG. 8 c shows the percent retention ofthe long-lasting oxyntomodulin conjugate relative to the start value.

MODE FOR INVENTION

Hereinafter, the present invention will be described in further detailwith reference to examples. It is to be understood, however, that theseexamples are for illustrative purposes only and are not intended tolimit the scope of the present invention.

Example 1 Synthesis of Oxyntomodulin and Oxyntomodulin Derivatives

In order to measure the stabilities of oxyntomodulin and oxyntomodulinderivatives in the liquid formulation of the present invention,oxyntomodulin derivatives having the amino acid sequences shown in Table1 below were synthesized.

TABLE 1 Oxyntomodulin and oxyntomodulin derivatives SEQ ID NO SequenceSEQ ID NO: 1 HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNR NNIA SEQ ID NO: 2CA-SQGTFTSDYSKYLDEEAVRLFIEWLMNTKR NRNNIA SEQ ID NO: 3CA-SQGTFTSDYSKYLDERRAQDFVAWLKNTGP SSGAPPPS SEQ ID NO: 4CA-GQGTFTSDYSRYLEEEAVRLFIEWLKNGGP SSGAPPPS SEQ ID NO: 5CA-GQGTFTSDYSRQMEEEAVRLFIEWLKNGGP SSGAPPPS SEQ ID NO: 6CA-GEGTFTSDLSRQMEEEAVRLFIEWAAHSQG TFTSDYSKYLD SEQ ID NO: 7CA-SQGTFTSDYSRYLDEEAVRLFIEWLMNTK SEQ ID NO: 8CA-SQGTFTSDLSRQLEEEAVRLFIEWLMNK SEQ ID NO: 9CA-GQGTFTSDYSRYLDEEAVXLFIEWLMNTKR NRNNIA SEQ ID NO: 10CA-SQGTFTSDYSRQMEEEAVRLFIEWLMNGGP SSGAPPPSK SEQ ID NO: 11CA-GEGTFTSDLSRQMEEEAVRLFIEWAAHSQG TFTSDYSRYLDK SEQ ID NO: 12CA-SQGTFTSDYSRYLDGGGHGEGTFTSDLSKQ MEEEAVK SEQ ID NO: 13CA-SQGTFTSDYSRYLDXEAVXLFIEWLMNTK SEQ ID NO: 14CA-GQGTFTSDYSRYLDEEAVXLFIXWLMNTKR NRNNIA SEQ ID NO: 15CA-GQGTFTSDYSRYLDEEAVRLFIXWLMNTKR NRNNIA SEQ ID NO: 16CA-SQGTFTSDLSRQLEGGGHSQGTFTSDLSRQ LEK SEQ ID NO: 17CA-SQGTFTSDYSRYLDEEAVRLFIEWIRNTKR NRNNIA SEQ ID NO: 18CA-SQGTFTSDYSRYLDEEAVRLFIEWIRNGGP SSGAPPPSK SEQ ID NO: 19CA-SQGTFTSDYSRYLD E EAV K LFIEWIRNTKR NRNNIA SEQ ID NO: 20CA-SQGTFTSDYSRYLD E EAV K LFIEWIRNGGP SSGAPPPSK SEQ ID NO: 21CA-SQGTFTSDYSRQLEEEAVRLFIEWVRNTKR NRNNIA SEQ ID NO: 22 DA-SQGTFTSDYSKYLDE KRA K EFVQWLMNTK SEQ ID NO: 23 HAibQGTFTSDYSKYLDEKRAKEFVCWLMNTSEQ ID NO: 24 HAibQGTFTSDY SKYLDEKRAK EFVQWLMNT C SEQ ID NO: 25HAibQGTFTSDYSKYLD E KRA K EFVQWLMNIC SEQ ID NO: 26 HAibQGTFTSDYS K YLD EKRAKEFVQWLMNTC SEQ ID NO: 27 HAibQGTFTSDYSKYLD E QAA K EFICWLMNTSEQ ID NO: 28 HAibQGTFTSDY SKYLDEK RAKEFVQWLMNT SEQ ID NO: 29H(d)SQGTFTSDYSKYLDSRRAQDFVQWLMNTK RNRNNIA SEQ ID NO: 30CA-SQGTFTSDYSKYLDSRRAQDFVQWLMNTKR NRNNIA SEQ ID NO: 31CA-(d)SQGTFTSDYSKYLDSRRAQDFVQWLMN TKRNRNNIA SEQ ID NO: 32CA-AibQGTFTSDYSKYLDEKRAKEFVQWLMNT C SEQ ID NO: 33HAibQGTFTSDYAKYLDEKRAKEFVQWLMNTC SEQ ID NO: 34YAibQGTFTSDYSKYLDEKRAKEFVQWLMNTC

In Table a above, the amino acid residues indicated by bold letters meanamino acids that form rings, and the amino acid residues indicated by Xmean alpha-methyl-glutamic acid that is a non-native amino acid. Inaddition, CA indicates 4-imidazoacetyl, DA indicates desamino-histidyl,and (d)S indicates d-serine.

Example 2 Preparation of Conjugate Comprising Oxyntomodulin Derivative(SEQ ID NO: 25) and Immunoglobulin Fc (Oxyntomodulin Derivative (SEQ IDNO: 25) Linked to Immunoglobulin Fc Region)

First, in order to PEGylate MAL-10K-ALD PEG at a cysteine residue atamino acid position 30 of an oxyntomodulin derivative (SEQ ID NO: 25),the oxyntomodulin derivative (SEQ ID NO: 25) and MAL-10K-ALD PEG wereallowed to react with each other at a molar ratio of 1:3 and a proteinconcentration of 3 mg/ml at room temperature for 3 hours. Herein, thereaction was performed in 50 mM Tris buffer (pH 8.0) in the presence of1M guanidine. After completion of the reaction, the reaction solutionwas applied to a SOURCE S column to purify an oxyntomodulin derivativemono-PEGylated at the cysteine (column: SOURCE S, flow rate: 2.0 ml/min,gradient: A 0→100% 50 min B (A: 20 mM Na-citrate, pH 3.0+45% ethanol, B:A+1M KCl)) (FIG. 1 a). FIG. 1 a is a graph showing the results obtainedby purifying the mono-PEGylated oxyntomodulin derivative (SEQ ID NO: 25)through the SOURCE S purification column.

Then, the purified mono-PEGylated oxyntomodulin derivative (SEQ ID NO:25) and an immunoglobulin Fc were reacted with each other at a molarratio of 1:5 and a protein concentration of 20 mg/ml at 4° C. for 16hours. The reaction was performed in 100 mM potassium phosphate buffer(pH 6.0) in the presence of 20 mM SCB as a reducing agent. Aftercompletion of the reaction, the reaction was applied to a SOURCE 15Qpurification column (column: SOURCE 15Q, flow rate: 2.0 ml/min,gradient: A 0→4% 1 min B→20% 80 min B (A: 20 mM Tris-HCl, pH 7.5, B:A+1M NaCl)) (FIG. 1 b) and a source ISO purification column (column:SOURCE ISO, flow rate: 2.0 ml/min, gradient: B 0→100% 100 min A (A: 20mM Tris-HCl, pH 7.5, B: A+1.1M AS)) (FIG. 1 c) to purify a conjugatecomprising the oxyntomodulin derivative (SEQ ID NO: 25) and theimmunoglobulin Fc. FIG. 1 b is a graph showing the results obtained bypurifying the conjugate, comprising the oxyntomodulin derivative (SEQ IDNO: 25) and the immunoglobulin Fc, through the SOURCE 15Q purificationcolumn, and FIG. 1 c is a graph showing the results obtained bypurifying the conjugate, comprising the oxyntomodulin derivative (SEQ IDNO: 25) and the immunoglobulin Fc, through the Source ISO purificationcolumn.

The oxyntomodulin conjugate prepared as described above was developed toincrease the blood half-life of oxyntomodulin. It comprises theimmunoglobulin Fc region, the non-peptidyl polymer and theoxyntomodulin, linked covalently to each other in a site-specificmanner, and has a significantly increased blood half-life.

Example 3 Evaluation of Stability of Long-Lasting OxyntomodulinConjugate According to pH

In order to evaluate the stability of the long-lasting oxyntomodulinconjugate (prepared in Example 2) in liquid formulations, thelong-lasting oxyntomodulin conjugate was stored in the compositionsshown in Table 2 at 25° C. for 0-2 weeks, and then was analyzed by ionexchange-high performance liquid chromatography (IE-HPLC), sizeexclusion-high performance liquid chromatography (SE-HPLC) and reversephase-high performance liquid chromatography (RP-HPLC). For storage ofthe oxyntomodulin conjugate, citrate buffer as buffer, mannitol as sugaralcohol, and polysorbate 20 as a nonionic surfactant were used. InTables 3, 4 and 5 below, IE-HPLC (%), SE-HPLC (%) and RP-HPLC (%)indicate area %/start area %, which indicates the percent retention ofthe long-lasting oxyntomodulin conjugate relative to the start value.Table 3 shows the IE-HPLC area (%) of the long-lasting oxyntomodulinconjugate after storage, Table 4 shows the SE-HPLC area (%) of thelong-lasting oxyntomodulin conjugate after storage, and Table 5 showsthe RP-HPLC area (%) of the long-lasting oxyntomodulin conjugate afterstorage.

TABLE 2 Long- Isotonic Sugar lasting pH Buffer agent alcohol SurfactantOthers oxyntomodulin #1 4.8 20 mM — 10% Mannitol 0.005% 0.1 mg/mL 10mg/mL Na-Citrate Polysorbate 20 Methionine #2 5.2 20 mM — 10% Mannitol0.005% 0.1 mg/mL 10 mg/mL Na-Citrate Polysorbate 20 Methionine #3 5.6 20mM — 10% Mannitol 0.005% 0.1 mg/mL 10 mg/mL Na-Citrate Polysorbate 20Methionine #4 6.0 20 mM — 10% Mannitol 0.005% 0.1 mg/mL 10 mg/mLNa-Citrate Polysorbate 20 Methionine #5 6.4 20 mM — 10% Mannitol 0.005%0.1 mg/mL 10 mg/mL Na-Citrate Polysorbate 20 Methionine

TABLE 3 IE-HPLC (%) #1 #2 #3 #4 #5 0 week 100.0 100.0 100.0 100.0 100.02 weeks 97.6 97.2 96.8 94.0 85.2

TABLE 4 SE-HPLC (%) #1 #2 #3 #4 #5 0 week 100.0 100.0 100.0 100.0 100.02 weeks 99.7 99.8 99.8 99.3 99.4

TABLE 5 RP-HPLC (%) #1 #2 #3 #4 #5 0 week 100.0 100.0 100.0 100.0 100.02 weeks 83.0 84.4 86.7 65.5 74.0

As can be seen from the results of IE-HPLC (%) in Table 3 above, theoxyntomodulin conjugate was more stable at lower pH. In the results ofSE-HPLC in Table 4, the oxyntomodulin conjugate was most stable at a pHof 5.2, and in the results of RP-HPLC in Table 5, the oxyntomodulinconjugate was most stable at a pH of 5.6. Although the stability at pHdid differ between the analysis methods, the difference in retentionbetween pHs was the greatest in the RP-HPLC analysis method. Thissuggests that the oxyntomodulin conjugate was most stable at a pH of5.6.

Example 4 Evaluation of Stability of Long-Lasting OxyntomodulinConjugate According to the Kind of Sugar Alcohol and the Presence orAbsence of Isotonic Agent

The present inventors tested the influences of the kind of sugar alcoholas a stabilizer and the presence or absence of sodium chloride as anisotonic agent on the stability of the long-lasting oxyntomodulinconjugate. Specifically, using the citrate buffer (pH 5.6) selected inExample 3, the long-lasting oxyntomodulin conjugate was stored in thecompositions shown in Table 6 below at 25° C. for 0-4 weeks, and thenwas analyzed by IE-HPLC, SE-HPLC and RP-HPLC. In Tables 7, 8 and 9below, IE-HPLC (%), SE-HPLC (%) and RP-HPLC (%) indicate area %/startarea %, which indicates the percent retention of the long-lastingoxyntomodulin conjugate relative to the start value. Table 7 shows theIE-HPLC area (%) of the long-lasting oxyntomodulin conjugate afterstorage, Table 8 shows the SE-HPLC area (%) of the long-lastingoxyntomodulin conjugate after storage, and Table 9 shows the RP-HPLCarea (%) of the long-lasting oxyntomodulin conjugate after storage.

TABLE 6 Isotonic Sugar Long-lasting pH Buffer agent alcohol SurfactantOthers oxyntomodulin #1 5.6 20 mM — 10% 0.005% — 10 mg/mL Na-CitrateMannitol Polysorbate 20 #2 5.6 20 mM — 10% 0.005% — 10 mg/mL Na-CitrateSorbitol Polysorbate 20 #3 5.6 20 mM — 10% 0.005% — 10 mg/mL Na-CitrateGlycerol Polysorbate 20 #4 5.6 20 mM 150 mM 10% 0.005% — 10 mg/mLNa-Citrate NaCl Mannitol Polysorbate 20

TABLE 7 IE-HPLC (%) #1 #2 #3 #4 0 week 100.0 100.0 100.0 100.0 1 week98.8 98.8 88.3 98.3 2 weeks 97.2 96.9 79.0 95.0 3 weeks 94.4 94.5 63.093.8 4 weeks 91.6 91.8 55.8 91.6

TABLE 8 SE-HPLC (%) #1 #2 #3 #4 0 week 100.0 100.0 100.0 100.0 1 week99.9 100.0 92.0 100.0 2 weeks 99.7 99.8 84.7 99.9 3 weeks 99.2 99.4 79.299.5 4 weeks 98.4 98.6 76.0 98.8

TABLE 9 RP-HPLC (%) #1 #2 #3 #4 0 week 100.0 100.0 100.0 100.0 1 week98.9 98.7 90.0 98.4 2 weeks 96.0 95.6 80.6 95.0 3 weeks 94.0 93.6 75.292.9 4 weeks 92.6 91.1 70.0 90.1

As can be seen in Tables 6 to 9 above, the long-lasting oxyntomodulinconjugate was more stable in mannitol or sorbitol than in glycerol atthe same concentration. The results of RP-HPLC indicated that thelong-lasting oxyntomodulin conjugate was a little stable in mannitolthan in sorbitol. In addition, the stability of the long-lastingoxyntomodulin conjugate did not significantly differ between thepresence and absence of sodium chloride as an isotonic agent.

Example 5 Evaluation of Stability of Long-Lasting OxyntomodulinConjugate According to Concentration of Sugar Alcohol

The present inventors tested the influence of the concentration of sugaralcohol as a stabilizer on the stability of the long-lastingoxyntomodulin conjugate. Specifically, using the citrate buffer (pH 5.6and mannitol selected in the above Examples, the long-lastingoxyntomodulin conjugate was stored in the compositions shown in Table 10below at 25° C. for 0-4 weeks, and then was analyzed by IE-HPLC, SE-HPLCand RP-HPLC. In Tables 11, 12 and 13 below, IE-HPLC (%), SE-HPLC (%) andRP-HPLC (%) indicate area %/start area %, which indicates the percentretention of the long-lasting oxyntomodulin conjugate relative to thestart value. Table 11 shows the IE-HPLC area (%) of the long-lastingoxyntomodulin conjugate after storage, Table 12 shows the SE-HPLC area(%) of the long-lasting oxyntomodulin conjugate after storage, and Table13 shows the RP-HPLC area (%) of the long-lasting oxyntomodulinconjugate after storage.

TABLE 10 Isotonic Sugar Long-lasting pH Buffer agent alcohol SurfactantOthers oxyntomodulin #1 5.6 20 mM —  2% Mannitol 0.005% — 10 mg/mLNa-Citrate Polysorbate 20 #2 5.6 20 mM —  5% Mannitol 0.005% — 10 mg/mLNa-Citrate Polysorbate 20 #3 5.6 20 mM — 10% Mannitol 0.005% — 10 mg/mLNa-Citrate Polysorbate 20 #4 5.6 20 mM — 15% Mannitol 0.005% — 10 mg/mLNa-Citrate Polysorbate 20

TABLE 11 IE-HPLC (%) #1 #2 #3 #4 0 week 100.0 100.0 100.0 100.0 1 week79.7 98.7 98.8 79.0 2 weeks 57.6 97.6 97.9 61.0 3 weeks 39.8 97.1 97.249.2 4 weeks 34.9 95.5 95.5 43.4

TABLE 12 SE-HPLC (%) #1 #2 #3 #4 0 week 100.0 100.0 100.0 100.0 1 week97.3 99.4 100.0 99.5 2 weeks 89.0 99.4 99.8 95.4 3 weeks 79.4 99.3 99.490.5 4 weeks 74.7 98.4 99.1 83.5

TABLE 13 RP-HPLC (%) #1 #2 #3 #4 0 week 100.0 100.0 100.0 100.0 1 week89.8 93.2 98.2 95.2 2 weeks 80.3 85.3 94.7 90.9 3 weeks 71.9 78.5 91.184.1 4 weeks 66.0 71.0 89.1 76.5

As can be seen in Tables 10 to 13, the long-lasting oxyntomodulinconjugate was stable in the presence of 5% mannitol or 10% mannitol.However, a protein precipitate was formed in the presence of 2% mannitolor 15% mannitol. The results of IE-HPLC or SE-HPLC indicated that thestability of the long-lasting oxyntomodulin conjugate was similarbetween 10% mannitol and 5% mannitol. The results of RP-HPLC indicatedthat the stability of the long-lasting oxyntomodulin conjugate was morestable in 10% mannitol than in 5% mannitol.

Example 6 Evaluation of Stability of Long-Lasting OxyntomodulinConjugate According to the Concentration of Surfactant and the Presenceor Absence of Amino Acid

The present inventors tested the influences of the concentration of asurfactant as a stabilizer and the presence or absence of an amino acidon the stability of the long-lasting oxyntomodulin conjugate. Using thecitrate buffer (pH 5.6) and citrate buffer and 10% mannitol selected inthe above Examples, the long-lasting oxyntomodulin conjugate was storedin the compositions shown in Table 14 below at 25° C. for 0-4 weeks, andthen was analyzed by IE-HPLC, SE-HPLC and RP-HPLC. In Tables 15, 16 and17 below, IE-HPLC (%), SE-HPLC (%) and RP-HPLC (%) indicate area %/startarea %, which indicates the percent retention of the long-lastingoxyntomodulin conjugate relative to the start value. Table 15 shows theIE-HPLC area (%) of the long-lasting oxyntomodulin conjugate afterstorage, Table 16 shows the SE-HPLC area (%) of the long-lastingoxyntomodulin conjugate after storage, and Table 17 shows the RP-HPLCarea (%) of the long-lasting oxyntomodulin conjugate after storage.

TABLE 14 Isotonic Sugar Long-lasting pH Buffer agent alcohol SurfactantOthers oxyntomodulin #1 5.6 20 mM — 10% 0.005% — 10 mg/mL Na-CitrateMannitol Polysorbate 20 #2 5.6 20 mM — 10% 0.02% — 10 mg/mL Na-CitrateMannitol Polysorbate 20 #3 5.6 20 mM — 10% 0.05% — 10 mg/mL Na-CitrateMannitol Polysorbate 20 #4 5.6 20 mM — 10% 0.02% 0.1 mg/ml 10 mg/mLNa-Citrate Mannitol Polysorbate 20 Methionine

TABLE 15 IE-HPLC (%) #1 #2 #3 #4 0 week 100.0 100.0 100.0 100.0 1 week96.0 96.5 96.4 96.2 2 weeks 95.1 94.7 95.1 95.3 3 weeks 92.7 92.0 92.292.7 4 weeks 89.7 89.1 89.2 89.7

TABLE 16 SE-HPLC (%) #1 #2 #3 #4 0 week 100.0 100.0 100.0 100.0 1 week99.4 99.7 99.7 99.6 2 weeks 99.3 99.5 99.2 99.3 3 weeks 98.3 98.1 98.399.1 4 weeks 97.3 97.1 97.3 97.9

TABLE 17 RP-HPLC (%) #1 #2 #3 #4 0 week 100.0 100.0 100.0 100.0 1 week97.3 97.8 98.1 97.4 2 weeks 79.2 79.2 77.5 79.7 3 weeks 71.5 71.6 69.573.3 4 weeks 65.1 65.0 65.0 67.8

As can be seen from the results in Tables 14 to 17, the long-lastingoxyntomodulin conjugate was most stable in the composition containing0.02% polysorbate 20 and 0.1 mg/mL methionine.

Example 7 Evaluation of Stability of Long-Lasting OxyntomodulinConjugate According to pH and the Concentration of Sugar Alcohol

The present inventors tested the influences of pH and the concentrationof sugar alcohol as a stabilizer on the stability of the long-lastingoxyntomodulin conjugate. Specifically, using the 0.02% polysorbate 20and 0.1 mg/mL methionine selected in the above Examples, thelong-lasting oxyntomodulin conjugate was stored in the compositionsshown in Table 18 below at 25° C. for 0-4 weeks, and then was analyzedby IE-HPLC, SE-HPLC and RP-HPLC. In Tables 19, 20 and 21 below, IE-HPLC(%), SE-HPLC (%) and RP-HPLC (%) indicate area %/start area %, whichindicates the percent retention of the long-lasting oxyntomodulinconjugate relative to the start value. Table 19 shows the IE-HPLC area(%) of the long-lasting oxyntomodulin conjugate after storage, Table 20shows the SE-HPLC area (%) of the long-lasting oxyntomodulin conjugateafter storage, and Table 21 shows the RP-HPLC area (%) of thelong-lasting oxyntomodulin conjugate after storage.

TABLE 18 Long- Isotonic Sugar lasting pH Buffer agent alcohol SurfactantOthers oxyntomodulin #1 5.2 20 mM —  5% 0.02% 0.1 mg/ml 10 mg/mLNa-Citrate Mannitol Polysorbate 20 Methionine #2 5.2 20 mM — 10% 0.02%0.1 mg/ml 10 mg/mL Na-Citrate Mannitol Polysorbate 20 Methionine #3 5.220 mM — 15% 0.02% 0.1 mg/ml 10 mg/mL Na-Citrate Mannitol Polysorbate 20Methionine #4 5.6 20 mM —  5% 0.02% 0.1 mg/ml 10 mg/mL Na-CitrateMannitol Polysorbate 20 Methionine #5 5.6 20 mM — 10% 0.02% 0.1 mg/ml 10mg/mL Na-Citrate Mannitol Polysorbate 20 Methionine #6 5.6 20 mM — 15%0.02% 0.1 mg/ml 10 mg/mL Na-Citrate Mannitol Polysorbate 20 Methionine#7 6.0 20 mM —  5% 0.02% 0.1 mg/ml 10 mg/mL Na-Citrate MannitolPolysorbate 20 Methionine #8 6.0 20 mM — 10% 0.02% 0.1 mg/ml 10 mg/mLNa-Citrate Mannitol Polysorbate 20 Methionine #9 6.0 20 mM — 15% 0.02%0.1 mg/ml 10 mg/mL Na-Citrate Mannitol Polysorbate 20 Methionine

TABLE 19 IE-HPLC (%) #1 #2 #3 #4 #5 #6 #7 #8 #9 0 week 100.0 100.0 100.0100.0 100.0 100.0 100.0 100.0 100.0 1 week 99.4 99.6 97.1 97.2 97.6 98.090.6 92.9 93.4 2 weeks 97.5 97.8 90.5 93.8 94.1 93.2 81.5 83.6 85.6 3weeks 93.0 93.6 82.6 86.0 86.9 87.1 71.2 74.6 77.0 4 weeks 90.0 90.571.1 85.0 85.6 85.7 62.7 66.3 68.9

TABLE 20 SE-HPLC (%) #1 #2 #3 #4 #5 #6 #7 #8 #9 0 week 100.0 100.0 100.0100.0 100.0 100.0 100.0 100.0 100.0 1 week 98.8 99.5 98.8 99.5 99.3 98.599.6 100.0 100.7 2 weeks 98.8 99.1 91.1 98.4 97.1 96.9 97.3 98.3 97.9 3weeks 97.7 98.3 96.4 98.1 98.4 98.2 98.1 99.1 100.8 4 weeks 98.0 98.594.9 97.9 98.3 98.3 97.6 98.2 99.0

TABLE 21 RP-HPLC (%) #1 #2 #3 #4 #5 #6 #7 #8 #9 0 week 100.0 100.0 100.0100.0 100.0 100.0 100.0 100.0 100.0 1 week 78.2 78.2 42.1 88.2 90.7 89.499.9 99.1 100.7 2 weeks 60.7 58.6 — 80.4 80.2 80.6 96.8 93.4 92.0 3weeks 47.5 41.9 — 79.4 77.3 69.8 97.6 92.8 93.6 4 weeks 34.8 28.0 — 72.865.0 62.6 96.9 91.1 88.9

As can be seen from the results in Tables above, the results of IE-HPLCindicated that the stability of the long-lasting oxyntomodulin conjugatewas higher in the order of pH 5.2, pH 5.6 and pH 6.0. The results ofRP-HPLC indicated that the stability of the long-lasting oxyntomodulinconjugate was higher in the order of pH 6.0, pH 5.6 and pH 5.2. Theresults of SE-HPLC indicated that the stability of the long-lastingoxyntomodulin conjugate did not significantly differ between pH 5.2, pH5.6 and pH 6.0. In other words, the results of IE-HPLC, RP-HPLC andSE-HPLC indicated that the long-lasting oxyntomodulin conjugate wasstable at pH 5.6.

Meanwhile, the results of IE-HPLC and SE-HPLC indicated that thelong-lasting oxyntomodulin conjugate did not significantly differbetween mannitol concentrations at pH 5.6. However, in the results ofRP-HPLC, the long-lasting oxyntomodulin conjugate was more stable in 5%mannitol than in 10% or 15% mannitol at pH 5.6.

Example 8 Evaluation of Stability of Long-Lasting OxyntomodulinConjugate According to pH and the Kind of Buffer

The present inventors tested the influences of pH and the kind of bufferas a stabilizer on the stability of the long-lasting oxyntomodulinconjugate. Specifically, using the 0.02% polysorbate 20, 0.1 mg/mLmethionine and 5% mannitol selected in the above Examples, thelong-lasting oxyntomodulin conjugate was stored in the compositionsshown in Table 22 below at 25° C. for 0-4 weeks, and then was analyzedby IE-HPLC, SE-HPLC and RP-HPLC.

In Tables 23, 24 and 25 below, IE-HPLC (%), SE-HPLC (%) and RP-HPLC (%)indicate area %/start area %, which indicates the percent retention ofthe long-lasting oxyntomodulin conjugate relative to the start value.Table 23 shows the IE-HPLC area (%) of the long-lasting oxyntomodulinconjugate after storage, Table 24 shows the SE-HPLC area (%) of thelong-lasting oxyntomodulin conjugate after storage, and Table 25 showsthe RP-HPLC area (%) of the long-lasting oxyntomodulin conjugate afterstorage.

TABLE 22 Isotonic Sugar Long-lasting pH Buffer agent alcohol SurfactantOthers oxyntomodulin #1 5.6 20 mM — 5% 0.02% 0.1 mg/ml 10 mg/mLNa-Citrate Mannitol Polysorbate 20 Methionine #2 5.8 20 mM — 5% 0.02%0.1 mg/ml 10 mg/mL Na-Citrate Mannitol Polysorbate 20 Methionine #3 5.820 mM — 5% 0.02% 0.1 mg/ml 10 mg/mL Na-Acetate Mannitol Polysorbate 20Methionine #4 5.8 10 mM — 5% 0.02% 0.1 mg/ml 10 mg/mL Histidine MannitolPolysorbate 20 Methionine #5 5.8 10 mM — 5% 0.02% 0.1 mg/ml 10 mg/mL Na-Mannitol Polysorbate 20 Methionine Phosphate

TABLE 23 IE-HPLC (%) #1 #2 #3 #4 #5 0 week 100.0 100.0 100.0 100.0 100.01 week 97.6 96.8 96.5 97.2 96.1 2 weeks 93.9 91.4 90.6 92.6 89.6 3 weeks90.4 88.1 86.9 89.2 84.4 4 weeks 90.1 87.0 84.8 87.4 81.7

TABLE 24 SE-HPLC (%) #1 #2 #3 #4 #5 0 week 100.0 100.0 100.0 100.0 100.01 week 99.4 99.8 99.8 99.7 99.7 2 weeks 100.5 100.2 100.0 100.0 99.3 3weeks 100.4 100.2 99.1 99.6 98.2 4 weeks 100.1 99.2 99.0 98.8 97.7

TABLE 25 RP-HPLC (%) #1 #2 #3 #4 #5 0 week 100.0 100.0 100.0 100.0 100.01 week 99.1 97.2 99.4 99.5 99.0 2 weeks 97.6 97.7 99.4 99.8 99.3 3 weeks96.1 98.7 97.2 98.9 97.4 4 weeks 98.0 96.6 97.6 98.3 98.1

As can be seen from the results in Tables 23 to 25, the results ofSE-HPLC or RP-HPLC indicated that the stability of the long-lastingoxyntomodulin conjugate did not significantly differ between pH 5.6 andpH 5.8. The results of IE-HPLC indicated that the long-lastingoxyntomodulin conjugate was more stable at pH 5.6 than at pH 5.8. Theresults of SE-HPLC showed that the stability of the long-lastingoxyntomodulin conjugate did not significantly differ between the buffersat the same pH. In addition, the results of IE-HPLC or RP-HPLC indicatedthat the long-lasting oxyntomodulin conjugate was most stable inhistidine at the same pH.

Example 9 Evaluation of Influences of the Presence or Absence ofPreservative and the Concentration of Long-Lasting OxyntomodulinConjugate on the Stability of Long-Lasting Oxyntomodulin Conjugate

The present inventors tested the influences of the presence or absenceof a preservative as a stabilizer and the concentration of thelong-lasting oxyntomodulin conjugate on the stability of thelong-lasting oxyntomodulin conjugate. Specifically, using histidinebuffer (pH 5.6), 0.02% polysorbate 20, 0.1 mg/mL methionine and 5%mannitol, selected in the above Examples, the long-lasting oxyntomodulinconjugate was stored in the compositions shown in Table 26 below at 25°C. for 0-4 weeks, and then was analyzed by IE-HPLC, SE-HPLC and RP-HPLC.In Tables 27, 28 and 29 below, IE-HPLC (%), SE-HPLC (%) and RP-HPLC (%)indicate area %/start area %, which indicates the percent retention ofthe long-lasting oxyntomodulin conjugate relative to the start value.Table 27 shows the IE-HPLC area (%) of the long-lasting oxyntomodulinconjugate after storage, Table 28 shows the SE-HPLC area (%) of thelong-lasting oxyntomodulin conjugate after storage, and Table 29 showsthe RP-HPLC area (%) of the long-lasting oxyntomodulin conjugate afterstorage.

TABLE 26 Isotonic Sugar Long-lasting pH Buffer agent alcohol SurfactantOthers oxyntomodulin #1 5.6 10 mM — 5% Mannitol 0.02% 0.1 mg/ml 10 mg/mLHistidine Polysorbate 20 Methionine #2 5.6 10 mM — 5% Mannitol 0.02% 0.1mg/ml 10 mg/mL Histidine Polysorbate 20 Methionine 0.27% m- cresol #35.6 10 mM — 5% Mannitol 0.02% 0.1 mg/ml 40 mg/mL Histidine Polysorbate20 Methionine #4 5.6 10 mM — 5% Mannitol 0.02% 0.1 mg/ml 40 mg/mLHistidine Polysorbate 20 Methionine 0.27% m- cresol

TABLE 27 IE-HPLC (%) #1 #2 #3 #4 0 week 100.0 100.0 100.0 100.0 1 week97.9 98.2 97.7 97.1 2 weeks 95.3 95.7 95.1 94.3 3 weeks 93.6 92.9 93.491.8 4 weeks 91.3 90.4 90.2 88.4

TABLE 28 SE-HPLC (%) #1 #2 #3 #4 0 week 100.0 100.0 100.0 100.0 1 week99.7 99.6 99.5 99.4 2 weeks 99.3 99.1 99.0 97.9 3 weeks 99.1 98.9 98.797.0 4 weeks 98.8 98.0 98.0 95.4

TABLE 29 RP-HPLC (%) #1 #2 #3 #4 0 week 100.0 100.0 100.0 100.0 1 week100.1 100.0 99.8 99.7 2 weeks 99.4 99.5 99.2 99.1 3 weeks 98.3 98.3 98.898.5 4 weeks 98.8 97.9 97.7 97.4

As can be seen in Tables 26 to 29, the results of IE-HPLC, SE-HPLC orRP-HPLC indicated that the stability of long-lasting oxyntomodulinconjugate did not change even in the presence of the preservative anddid not differ according to the concentration thereof.

Although the preferred embodiments of the present invention have beendescribed for illustrative purposes, those skilled in the art willappreciate that various modifications, additions and substitutions arepossible, without departing from the scope and spirit of the inventionas disclosed in the accompanying claims.

1-31. (canceled)
 32. A liquid formulation of a long-lastingoxyntomodulin conjugate, comprising a pharmacologically active amount ofa long-lasting oxyntomodulin conjugate wherein an oxyntomodulin, whichis a physiologically active peptide, is linked to an immunoglobulin Fcregion; and an albumin-free stabilizer, wherein the stabilizer containsa buffer, a sugar alcohol and a nonionic surfactant.
 33. The liquidformulation of a long-lasting oxyntomodulin conjugate according to claim32, wherein the stabilizer further contains one or more selected fromthe group consisting of isotonic agents, sugars, polyhydric alcohols andamino acids.
 34. The liquid formulation of a long-lasting oxyntomodulinconjugate according to claim 32, wherein the oxyntomodulin is a peptidemade from pre-glucagon, a precursor of glucagon, and has an amino acidsequence of SEQ ID NO:
 1. 35. The liquid formulation of a long-lastingoxyntomodulin conjugate according to claim 32, wherein the oxyntomodulinis native oxyntomodulin or a precursor, derivative, fragment or variantthereof.
 36. The liquid formulation of a long-lasting oxyntomodulinconjugate according to claim 35, wherein the derivative has any oneamino acid sequence selected from the group consisting of SEQ ID NOS: 2to
 34. 37. The liquid formulation of a long-lasting oxyntomodulinconjugate according to claim 32, wherein the conjugate is prepared byusing a non-peptidyl polymer or recombination technique.
 38. The liquidformulation of a long-lasting oxyntomodulin conjugate according to claim37, wherein the non-peptidyl polymer is selected the group consisting ofpolypropylene glycol, an ethylene glycol/propylene glycol copolymer,polyoxyethylated polyol, polyvinyl alcohol, polysaccharides, dextran,polyvinyl ethyl ether, biodegradable polymers, including polylactic acid(PLA) and polylactic-glycolic acid (PLGA); lipid polymers; chitins;hyaluronic acid; and combinations thereof.
 39. The liquid formulation ofa long-lasting oxyntomodulin conjugate according to claim 37, whereinthe non-peptidyl polymer is polyethylene glycol.
 40. The liquidformulation of a long-lasting oxyntomodulin conjugate according to claim32, wherein the sugar alcohol is one or more selected from the groupconsisting of mannitol, sorbitol and glycerol.
 41. The liquidformulation of a long-lasting oxyntomodulin conjugate according to claim40, wherein the concentration of the sugar alcohol in the liquidformulation is 2-15% (w/v).
 42. The liquid formulation of a long-lastingoxyntomodulin conjugate according to claim 32, wherein the buffer is oneor more selected from the group consisting of citrate, acetate,histidine and phosphate buffer.
 43. The liquid formulation of along-lasting oxyntomodulin conjugate according to claim 32, wherein thebuffer has a pH ranging from 4.5 to 7.0.
 44. The liquid formulation of along-lasting oxyntomodulin conjugate according to claim 33, wherein theisotonic agent is sodium chloride.
 45. The liquid formulation of along-lasting oxyntomodulin conjugate according to claim 32, wherein thenonionic surfactant is polysorbate or poloxamer.
 46. The liquidformulation of a long-lasting oxyntomodulin conjugate according to claim45, wherein the concentration of the nonionic surfactant in the liquidformulation is 0.001-0.1% (w/v).
 47. The liquid formulation of along-lasting oxyntomodulin conjugate according to claim 33, wherein theamino acid is methionine.
 48. The liquid formulation of a long-lastingoxyntomodulin conjugate according to claim 32, wherein the stabilizercontains a buffer having a pH ranging from 4.8 to 6.0, one or more sugaralcohols selected from the group consisting of mannitol and sorbitol,and polysorbate
 20. 49. The liquid formulation of a long-lastingoxyntomodulin conjugate according to claim 32, further comprising one ormore preservatives selected from the group consisting of m-cresol,phenol and benzyl alcohol.
 50. The liquid formulation of a long-lastingoxyntomodulin conjugate according to claim 49, wherein the concentrationof the preservative in the liquid formulation is 0.001-1% (w/v).
 51. Aliquid formulation of a long-lasting oxyntomodulin conjugate, comprisinga pharmacologically effective amount of a long-lasting oxyntomodulinconjugate wherein an oxyntomodulin, which is a physiologically activepeptide, is linked to an immunoglobulin Fc region; 5-50 mM histidine;2-15% (w/v) of mannitol; 0.01-1 mg/mL of methionine; and 0.001-0.1%(w/v) of polysorbate
 20. 52. A method for preparing the liquidformulation of claim 32, the method comprising the steps of: a)preparing a long-lasting oxyntomodulin conjugate; and b) mixing thelong-lasting oxyntomodulin conjugate, prepared in step a), with astabilizer containing a buffer, a sugar alcohol and a nonionicsurfactant.
 53. A method for preparing the liquid formulation of claim49, the method comprising the steps of: a) preparing a long-lastingoxyntomodulin conjugate; and b) mixing the long-lasting oxyntomodulinconjugate, prepared in step a), with a stabilizer, which contains abuffer, a sugar alcohol and a nonionic surfactant, and a preservative.54. The method of claim 52, wherein the stabilizer further contains oneor more selected from the group consisting of isotonic agents, sugars,polyhydric alcohols and amino acids.
 55. The method of claim 53, whereinthe stabilizer further contains one or more selected from the groupconsisting of isotonic agents, sugars, polyhydric alcohols and aminoacids.
 56. The liquid formulation of a long-lasting oxyntomodulinconjugate according to claim 32, wherein the immunoglobulin Fc region isa human aglycosylated IgG4 Fc region.